Targeting Angiogenesis in Cancer: Bevacizumab-Induced Platelet Activation as a Possible Cause for Unexpected Arterial Thromboembolic Events in Clinical Trials.

The humanized monoclonal VEGF antibody, bevacizumab (Avastin, Genentech), is approved in combination with standard chemotherapy for first-line treatment of patients with metastatic colorectal cancer (CRC) and also shows promising efficacy as anti-angiogenic immunotherapy in patients with non-small-cell lung cancer (NSCLC). A pooled analysis of five randomized, controlled trials (n=1745) showed that, compared to placebo, bevacizumab was associated with an increased risk of arterial thromboembolic events, especially in patients 65 years of age and older (8.5% vs. 2.9%, P<0.01). Because platelets play a crucial role in arterial thrombosis, we hypothesized that bevacizumab has direct platelet-stimulating activity. In a washed platelet system, bevacizumab alone had no effect on platelet aggregation. However, when combined with heparin (0.3 U/ml) and recombinant human VEGF (rhVEGF₁₆₅ — a homodimeric protein with heparin binding sites) in a 1:2 molar ratio of antibody to antigen to allow optimal formation of immune complexes (ICs) in solution, bevacizumab potently induced platelet aggregation (up to 80–100%; n=5). Bevacizumab-induced platelet aggregation (BIPA) was functionally dependent on Fc domain binding to the platelet low-affinity IgG receptor, FcγRIIA, as demonstrated by an inhibitory monoclonal CD32 antibody (IV.3). BIPA was potentiated in platelets pre-sensitized with low concentrations of ADP or epinephrine. In contrast, BIPA was virtually absent at excess concentrations of heparin (100 U/ml), suggesting that translocation of ICs to the platelet surface via available heparin binding sites (on platelets) was crucial for this platelet response. Unfractionated heparin and the low-molecular-weight heparin, enoxaparin, were equally effective in promoting BIPA. In a manner similar to heparin-PF4 antibodies from patients with heparin-induced thrombocytopenia (HIT), bevacizumab-rhVEGF₁₆₅-heparin ICs induced significant FcγRIIA-dependent dense granule release (>80%) in a ¹⁴C-serotonin release assay (SRA). While strong platelet responses were observed in both SRA and aggregometry, in which platelets were subjected to constant movement and low shear forces, respectively, bevacizumab-rhVEGF₁₆₅-heparin ICs had only negligible effects on platelet CD62P expression under static conditions, indicating a critical role for platelet-platelet contacts in bevacizumab-mediated FcγRIIA signaling. In summary, our results suggest bevacizumab can induce strong FcγRIIA-dependent platelet activation in vitro when complexed with rhVEGF₁₆₅ and heparin in an optimal stoichiometry. Due to their analogy to the pathomechanism of HIT, an acquired IgG-mediated disorder potentially associated with deleterious thrombosis, these findings may have direct clinical implications for older cancer patients with cardiovascular comorbidity, especially considering that many patients receive low doses of heparin for thromboprophylaxis and that elevated serum VEGF levels have been demonstrated in various types of malignancy, including CRC and NSCLC.